scholarly journals Identification of general acid catalyst for the ATPase activity of Lynch syndrome-related MutL homologs

2021 ◽  
Author(s):  
Kenji Fukui ◽  
Yuki Fujii ◽  
Takato Yano
Keyword(s):  
Lynch Syndrome ◽  
Atpase Activity ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Acid Catalyst ◽  
Base Catalysis ◽  
Mechanism Analysis ◽  
Missense Mutations ◽  
Aquifex Aeolicus ◽  
General Acid

Mutations of mismatch repair MutL homologs are causative of a hereditary cancer, Lynch syndrome. Investigation of MutL facilitates genetic diagnoses essential for cancer risk managements and therapies. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although existence of a general acid catalyst had not been conceived in the mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is accelerated by general acid-base catalysis. Analyses of mutant aqMutLs showed that Lys79 is the general acid, and the corresponding residues were confirmed to be critical for activities of human homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations.

The mechanism of the nonenzymatic iodination of tyrosine by molecular iodine

1988 ◽  
Vol 66 (9) ◽  
pp. 967-978 ◽  
Author(s):  
H. Brian Dunford ◽  
Adejare J. Adeniran
Keyword(s):  
Ph Dependence ◽  
Acid Catalyst ◽  
Order Rate Constant ◽  
Molecular Iodine ◽  
Base Catalysis ◽  
Slow Step ◽  
Rate Law ◽  
General Acid ◽  
Phenolic Group ◽  
Ph Range

Over the pH range 7–10, at very low buffer concentration, the nonenzymatic iodination of tyrosine obeys the rate law[Formula: see text]where kapp is the measured second order rate constant based upon the total initial concentrations of molecular iodine and tyrosine and K2 (units M) is the equilibrium constant for [Formula: see text]. The value of k′ is 3.5 × 10−8 M∙s−1. There are three plausible mechanisms that fit the experimental data. One, the simplest, is a concerted process in which hypoiodous acid attacks tyrosine with its phenolic group unionized. The other two involve the formation of an iodinated quinoid reactive intermediate species in a rapid pre-equilibrium between unionized tyrosine and either hypoiodous acid or molecular iodine. The pre-equilibrium, if it occurs, favors the initial reactants. It is followed by a slow step in which the quinoid is converted to mono-iodinated tyrosine. Positive deviations from the rate law for pH dependence indicate that some specific acid catalysis (H3O+) is occurring in the pH range 5–7. In the presence of sufficient buffer, general acid–base catalysis is observed with acetic acid acting as a general acid catalyst in the vicinity of pH 5 and carbonate acting as a general base at pH ~ 9.5. The nonenzymatic iodination of tyrosine occurs more rapidly as the pH is increased, in marked contrast to the peroxidase-catalyzed iodination, which has its optimum at low pH.

scholarly journals pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides

1971 ◽  
Vol 124 (1) ◽  
pp. 117-122 ◽  
Author(s):  
G. Lowe ◽  
Y. Yuthavong
Keyword(s):  
Acid Catalysis ◽  
Ph Dependence ◽  
Base Catalysis ◽  
General Base ◽  
Structure Activity ◽  
Equilibrium Binding ◽  
General Acid ◽  
Leaving Group ◽  
Hydrolysis Of ◽  
Equilibrium Binding Constant

The pH-dependence of the Michaelis–Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, Ks, is independent of pH between 3.7 and 9.3, whereas the acylation constant, k+2, shows bell-shaped pH-dependence with apparent pKa values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett ρ value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett ρ values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.

scholarly journals Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

2021 ◽  
Author(s):  
Lee M Stunkard ◽  
Aaron B Benjamin ◽  
James Bower ◽  
Tyler Huth ◽  
Jeremy Lohman
Keyword(s):  
Transition State ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Streptomyces Coelicolor ◽  
Base Catalysis ◽  
Acid Base ◽  
Transition State Analog ◽  
Catalytic Residues ◽  
General Acid

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

scholarly journals A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL

2020 ◽  
Vol 295 (33) ◽  
pp. 11643-11655
Author(s):  
Keisuke Izuhara ◽  
Kenji Fukui ◽  
Takeshi Murakawa ◽  
Seiki Baba ◽  
Takashi Kumasaka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Lynch Syndrome ◽  
Atpase Activity ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Aquifex Aeolicus ◽  
Dna Mismatch ◽  
Genes Encoding

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69–1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

scholarly journals Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

2021 ◽  
Author(s):  
Lee M Stunkard ◽  
Aaron B Benjamin ◽  
James Bower ◽  
Tyler Huth ◽  
Jeremy Lohman
Keyword(s):  
Transition State ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Streptomyces Coelicolor ◽  
Base Catalysis ◽  
Acid Base ◽  
Transition State Analog ◽  
Catalytic Residues ◽  
General Acid

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

2019 ◽  
Author(s):  
Adrian Roitberg ◽  
Pancham Lal Gupta
Keyword(s):  
Transfer Reaction ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Ph Effects ◽  
Dependent Manner ◽  
E Coli ◽  
Gar Tfase ◽  
Activity Curve ◽  
Ph Dependent ◽  
Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

scholarly journals pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

scholarly journals Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase

2020 ◽  
Author(s):  
Konstantin Laun ◽  
Iuliia Baranova ◽  
Jifu Duan ◽  
Leonie Kertess ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
H2 Evolution ◽  
Proton Reduction ◽  
Ph Values ◽  
Site Selective ◽  
H2 Oxidation ◽  
Diiron Site

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high oxidation activities, the majority of [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands that facilitate catalysis at low overpotential. Distinct proton transfer pathways connect the active site niche with the solvent, resulting in a non-trivial dependence of hydrogen turnover and bulk pH. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the natural pH dependence of hydrogen turnover as catalyzed by [FeFe]-hydrogenase.<br>

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